We hypothesize that long-term alcohol abuse results in changes in gene expression in brain and that these changes are responsible, at least partly, for alcohol dependence. We also hypothesize that there is significant structural variation in expression patterns of specific genes that can be detected by next generation sequencing. The goal of this project is to utilize this state-of-the-art technology to identify novel transcripts that are differentially expressed in brain regions Involved in drug reward of well-characterized human subjects as a function of alcohol intake, abuse, and dependence. To date, there are no published reports of gene expression in the human extended amygdala and related cortical projection regions. The results of these studies will identify as-yet-unknown alcohol responsive genes, including low abundance, splice variants, and expressed SNPs. In collaboration with other INIA investigators, we will have the opportunity to analyze and compare sequence data obtained from human, macaque, and rodents following alcohol exposure. The proposed experiments are a direct outcome of a prior INIA West Pilot Project awarded to the PI. Three Specific Aims are proposed: Aim 1 will test the hypothesis that next generation transcriptome profiling (RNA-seq) will identify differentially expressed RNAs in select brain regions of well-characterized alcohol-dependent cases compared to matched controls. Subsequent RT-PCR analysis will validate and define which of these are involved in mediating the effects of alcohol consumption. Aim 2 will test the hypothesis that next generation sequencing will identify unique small RNAs in human prefrontal cortex and amygdala. This will provide definitive information about the presence and brain regional distribution of these small RNA regulatory molecules, which will not only be of general importance in neuroscience, but will advance a molecular classification of alcohol dependence in key regions of the brain. Aim 3 will test the hypothesis that next generation sequencing will reveal novel expression changes in functional groups of genes found in the prefrontal cortex and amygdala of human alcoholics and changes will be seen in homologous brain regions using mouse and macaque models of excessive alcohol consumption. To address similarities and differences in transcriptome changes found in animal models of excessive alcohol consumption and human alcoholics across INIA sites, we will utilize differential expression and Weighted Gene Covariance Network Analysis (WGCNA). PUBLIC HEALTH RELEVANCE: We propose that alcohol-induced changes in brain function are due to alterations in gene expression and we will explore these changes with several innovative approaches to alcohol research, including next generation sequencing of the human brain transcriptome, identification of novel miRNAs, and network analysis of significantly changed genes. This work will provide new opportunities for gene-based diagnosis and treatment of alcohol dependence.